PyroMark PCR Kit

For PCR amplification of template DNA optimized for Pyrosequencing analysis

Highly specific amplification of bisulfite converted DNA and gDNA

Kit optimized for successful Pyrosequencing analysis

High yields of PCR product and no need for optimization

Convenient master mix with room-temperature setup

Optimized protocols

The PyroMark PCR Kit is specifically optimized for Pyrosequencing analysis, providing highly specific and unbiased amplification of template DNA for various applications, such as mutation detection, SNP analysis, methylation analysis, and sequencing. The kit is provided in a convenient master mix format consisting of HotStarTaq DNA Polymerase and optimized PyroMark Reaction Buffer for highly specific amplification. In addition, Q-Solution, a novel additive that enables efficient amplification of "difficult" (e.g., GC-rich) templates, is provided. The PyroMark PCR Kit is also supplied with CoralLoad Concentrate, which is strongly recommended for use with the PyroMark PCR Master Mix, for highly specific PCR and high yields of amplified DNA. CoralLoad Concentrate contains 2 gel-tracking dyes, allowing PCR products to be loaded directly on a gel and checked prior to Pyrosequencing.

[A] CoralLoad Concentrate contains [B] 2 gel-tracking dyes, enabling immediate gel loading of PCR products for easy visualization of DNA migration.|The PyroMark PCR Kit ensures greater amplification efficiency with high specificity. This results in [A] higher Pyrogram peaks and more reliable Pyrosequencing results, compared with [B] standard PCR.|Template DNA (10 ng) was amplified with the PyroMark PCR Kit in parallel with PCR master mixes from Suppliers R, I, and AII. Primers specific for Cyclin A1, BARHL1, MINT1, or DNMT3b were designed with the PyroMark Assay Design Software 2.0, and an annealing temperature gradient ranging from 48.4°C to 60.1°C was used. The PyroMark PCR Kit consistently produced higher amplicon yields and fewer nonspecific PCR products than the other master mixes, ensuring reliable results from downstream Pyrosequencing analyses. M: 50 bp ladder|K+ binds to the phosphate groups (P) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing principally the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains the high ratio of specific to nonspecific primer-template binding over a wide temperature range.|

PyroMark PCR Master Mix

The PyroMark PCR Kit enables PCR amplification of template DNA optimized for Pyrosequencing analysis. The convenient master mix format enables specific amplification of bisulfite converted DNA or genomic DNA from various starting materials, using only one protocol. Bisulfite treatment of genomic DNA converts nonmethylated cytosines to uracils and produces DNA that mainly consists of three bases. This less complex DNA is a challenging PCR template — the PCR products obtained are likely to have low yields, and nonspecific products might also be generated due to increased probability for mispriming. The balanced combination of salts in the PyroMark PCR Kit's unique master mix ensures specifically targeted primer binding and prevents mispriming. The special formulation also prevents accumulation of excess biotinylated primer and minimizes generation of artifacts that can interfere with the Pyrosequencing reaction. The high yields of specific PCR products obtained ensure reliable Pyrosequencing results.

During the annealing step of every PCR cycle, the buffer composition ensures a high ratio of specific-to-nonspecific primer binding (see figure "Increased specificity of primer annealing"). Owing to a uniquely balanced combination of KCl and (NH4)2SO4, the PCR buffer provides stringent primer annealing conditions over a wider range of annealing temperatures and Mg2+ concentrations, compared with conventional PCR buffers. Optimizing PCR by varying the annealing temperature or Mg2+ concentration is usually not required.

HotStarTaq DNA Polymerase

HotStarTaq DNA Polymerase, provided in the master mix, is a modified form of the recombinant 94 kDa Taq DNA Polymerase from QIAGEN. HotStarTaq DNA Polymerase is provided in an inactive state with no polymerase activity at ambient temperatures. This prevents the formation of misprimed products and primer–dimers at low temperatures. HotStarTaq DNA Polymerase is activated by a 15 minute, 95°C incubation step, that can easily be incorporated into existing thermal cycling programs. HotStarTaq DNA Polymerase provides high PCR specificity and often increases the yield of the specific PCR product. PCR setup is quick and convenient, since all reaction components can be combined at room temperature.

Q-Solution

The PyroMark PCR Master Mix is provided with Q-Solution, an innovative PCR additive that facilitates amplification of difficult templates by modifying the melting behavior of DNA. This unique reagent often enables or improves PCR caused by difficult templates (e.g., templates with a high degree of secondary structure or templates that are GC-rich). Unlike other commonly used PCR additives, such as DMSO, Q-Solution is used at just one working concentration, is nontoxic, and PCR purity is guaranteed. PCR fragments amplified in the presence of Q-Solution can be successfully analyzed by Pyrosequencing.

CoralLoad Concentrate

The kit is also supplied with CoralLoad Concentrate, which contains a gel loading reagent and 2 gel tracking dyes (see figure "CoralLoad Concentrate"). When using CoralLoad Concentrate, the PCR products can be directly loaded onto an agarose gel without prior addition of loading buffer. CoralLoad Concentrate can be added to the PCR without affecting amplification sensitivity or specificity. PCR fragments amplified in the presence of CoralLoad Concentrate can be successfully analyzed by Pyrosequencing.

Procedure

The reaction composition and cycling conditions are optimized for amplifying template DNA for Pyrosequencing analysis. Template DNA is combined with the convenient PyroMark PCR Mastermix, along with Q-Solution, CoralLoad concentrate, and amplification primers (one of which is biotinylated). HotStarTaq DNA Polymerase is inactive at room temperature, so it is not necessary to keep the reactions on ice during setup.

Applications

Pyrosequencing is increasingly important for research applications in a variety of disciplines. Pyrosequencing enables powerful and versatile analysis of genetic and epigenetic variation, whether examining drug-resistance development in pathogens, the role of epigenetic DNA methylation in gene expression regulation, genetic markers for specific phenotypes in livestock, or polymorphisms in forensic samples of mitochondrial DNA. The PyroMark PCR Kit provides the reagents necessary for reliable and highly specific template amplification for Pyrosequencing analysis.